Direct detection and characterization of foot-and-mouth disease virus in East Africa using a field-ready real-time PCR platform.

Effective control and monitoring of foot-and-mouth disease (FMD) relies upon rapid and accurate disease confirmation. Currently, clinical samples are usually tested in reference laboratories using standardized assays recommended by The World Organisation for Animal Health (OIE). However, the requirements for prompt and serotype-specific diagnosis during FMD outbreaks, and the need to establish robust laboratory testing capacity in FMD-endemic countries have motivated the development of simple diagnostic platforms to support local decision-making. Using a portable thermocycler, the T-COR™ 8, this study describes the laboratory and field evaluation of a commercially available, lyophilized pan-serotype-specific real-time RT-PCR (rRT-PCR) assay and a newly available FMD virus (FMDV) typing assay (East Africa-specific for serotypes: O, A, Southern African Territories [SAT] 1 and 2). Analytical sensitivity, diagnostic sensitivity and specificity of the pan-serotype-specific lyophilized assay were comparable to that of an OIE-recommended laboratory-based rRT-PCR (determined using a panel of 57 FMDV-positive samples and six non-FMDV vesicular disease samples for differential diagnosis). The FMDV-typing assay was able to correctly identify the serotype of 33/36 FMDV-positive samples (no cross-reactivity between serotypes was evident). Furthermore, the assays were able to accurately detect and type FMDV RNA in multiple sample types, including epithelial tissue suspensions, serum, oesophageal-pharyngeal (OP) fluid and oral swabs, both with and without the use of nucleic acid extraction. When deployed in laboratory and field settings in Tanzania, Kenya and Ethiopia, both assays reliably detected and serotyped FMDV RNA in samples (n = 144) collected from pre-clinical, clinical and clinically recovered cattle. These data support the use of field-ready rRT-PCR platforms in endemic settings for simple, highly sensitive and rapid detection and/or characterization of FMDV.

Evaluation of Two Lyophilized Molecular Assays to Rapidly Detect Foot-and-Mouth Disease Virus Directly from Clinical Samples in Field Settings.

Accurate, timely diagnosis is essential for the control, monitoring and eradication of foot-and-mouth disease (FMD). Clinical samples from suspect cases are normally tested at reference laboratories. However, transport of samples to these centralized facilities can be a lengthy process that can impose delays on critical decision making. These concerns have motivated work to evaluate simple-to-use technologies, including molecular-based diagnostic platforms, that can be deployed closer to suspect cases of FMD. In this context, FMD virus (FMDV)-specific reverse transcription loop-mediated isothermal amplification (RT-LAMP) and real-time RT-PCR (rRT-PCR) assays, compatible with simple sample preparation methods and in situ visualization, have been developed which share equivalent analytical sensitivity with laboratory-based rRT-PCR. However, the lack of robust 'ready-to-use kits' that utilize stabilized reagents limits the deployment of these tests into field settings. To address this gap, this study describes the performance of lyophilized rRT-PCR and RT-LAMP assays to detect FMDV. Both of these assays are compatible with the use of fluorescence to monitor amplification in real-time, and for the RT-LAMP assays end point detection could also be achieved using molecular lateral flow devices. Lyophilization of reagents did not adversely affect the performance of the assays. Importantly, when these assays were deployed into challenging laboratory and field settings within East Africa they proved to be reliable in their ability to detect FMDV in a range of clinical samples from acutely infected as well as convalescent cattle. These data support the use of highly sensitive molecular assays into field settings for simple and rapid detection of FMDV.

Epidemiological analysis of an outbreak of foot-and-mouth disease (serotype SAT2) on a large dairy farm in Kenya using regular vaccination.

During August-September 2012, an outbreak of Foot-and-mouth Disease (FMD) due to serotype Southern African Territories-2 (SAT2) occurred on a large, extensively grazed dairy farm in Nakuru County, Kenya. Over 29 days, 400/644 (62.1%) cattle were recorded as displaying clinical signs consistent with FMD. Out of the 18 management groups present, 17 had clinical cases (weighted mean incidence rate 3.5 per 100 cattle-days, 95% CI 2.4, 5.1; range 0.064-10.9). Transmission may have been encouraged when an infected group was moved to a designated isolation paddock. A four to five day minimum incubation period was apparent in five groups for which a point source exposure was evident. Further transmission was associated with the movement of individual animals incubating infection, use of a common dip and milking parlour, and grazing of susceptible groups in paddocks neighbouring to infectious cases. Animals over 18 months old appeared to be at highest risk of disease possibly due to milder clinical signs seen among younger animals resulting in reduced transmission or cases not being recorded. Cows with a breeding pedigree containing a greater proportion of zebu appeared to be at lower risk of disease. The outbreak occurred despite regular vaccination (three times per year) last performed approximately three months before the index case. Incidence risk by the lifetime number of doses received indicated limited or no vaccine effectiveness against clinical disease. Reasons for poor vaccine effectiveness are discussed with antigenic diversity of the SAT2 serotype and poor match between the field and vaccine strain as a likely explanation. Detailed field-derived epidemiological data based on individual animals are rarely presented in the literature for FMD, particularly in East-Africa and with the SAT2 serotype. This study provides a detailed account and therefore provides a greater understanding of FMD outbreaks in this setting. Additionally, this is the first study to provide field-derived evidence of poor vaccine effectiveness using a SAT2 vaccine. Further field-based measures of vaccine effectiveness in line with evaluation of human vaccines are needed to inform FMD control policy which has previously relied heavily upon experimental data and anecdotal experience.